A new family of assays for detecting potential chemical carcinogens is described. These assays measure the extent to which a substance alters the specificity of base selection during DNA replication in vitro. The basis of this measure is the incorporation into DNA of an analogue substrate relative to a natural substrate in a DNA polymerase reaction. The reaction utilizes a mammalian DNA polymerase, DNA templates from mammalian cells and analogue/natural competitive substrate pairs under conditions which normally select against analogue incorporation. Substances are tested for their ability to enhance the relative incorporation of the analogue substrate. Enhancement is taken as evidence that selection specificity has been altered, i.e. error-prone synthesis has been induced. Substances which do not require metabolic activation are tested for their primary site of action by pretreating the polymerase, by pretreating the template or by direct addition to the reaction. In addition, substances that require metabolic activation to interact with DNA are tested for the extent to which these interactions promote error-prone replication by using DNA templates extracted from tissues exposed to such substances. For example, using the incorporation of araATP relative to dATP in the presence of various heavy metal salts, we have found correlation between the increased ratios of analogue incorporation and the carcinogenicity of these metallic cations. Other analogue substrates, corresponding to each of the four normal substrates, are proposed for testing. The effects on each of these systems of treating either the DNA polymerase or the DNA template with these direct-acting carcinogens will be determined and, in addition, the sensitivity of the assays in detecting altered template activity for DNA extracted from cells exposed to two carcinogens that require metabolic activation will be established. These results will provide the basis for selecting and standardizing assays appropriate for use in screening potential environmental carcinogens.